The ends of mammalian chromosomes terminate in long arrays of TTAGGG repeats that have associated with them specific DNA binding proteins. These nucleoprotein complexes are known as telomeres and they function to preserve the integrity of chromosomes during the cell cycle by allowing the proper segregation during cell division. More specifically, telomeres shield the chromosome ends from degradation or end-on fusion, prevent the activation of DNA damage checkpoints, and modulate activity of telomerase, an enzyme that maintains the length of telomeres. Without this enzymatic activity, telomeres shorten at each cell division because the replication machinery fails to replicate DNA ends.
Human somatic cell chromosomes undergo normal telomere shortening at each cell division, and it is believed that this process is a tumor suppression mechanism that limits the number of potential replication cycles of the cell. However, in some cancers it has been demonstrated that telomeres do not undergo replication-associated shortening resulting in the transformed phenotype and subsequent tumorigenesis. On the other hand, the complete loss of telomeric DNA in tumors has also been observed, collectively suggesting that any modification to telomere length homeostasis can contribute to the carcinogenic phenotype. For this reason, much effort is currently focused on the characterization of factors responsible for maintaining the normal status of telomeres within the cell (Muniyappa and Kironmai, Crit. Rev. Biochem. Mol. Biol., 1998, 33, 297-336).
Telomeric repeat binding factor 2 (also known as TRF2, TERF2 and TRBF2) is one of two distinct telomeric DNA-binding proteins that are found associated with double-stranded telomeric repeat arrays in interphase and metaphase cells (the other being TRF1) (Broccoli et al., Nat. Genet., 1997, 17, 231-235). The protein exists as a homodimer, and contains a very basic N-terminus which is thought to modulate the DNA binding affinity of the protein (Broccoli et al., Nat. Genet., 1997, 17, 231-235). Telomeric repeat binding factor 2 is a ubiquitously expressed protein that contributes to the maintenance of telomere length by preventing end-to-end fusion of chromosomes (van Steensel et al., Cell, 1998, 92, 401-413). Griffith et al. have recently shown that telomeric repeat binding factor 2 functions to protect chromosome ends by remodeling the linear telomeres into t-loops, structures comprised of double stranded DNA (Griffith et al., Cell, 1999, 97, 503-514).
Functionalization studies in HeLa cells overexpressing intact and truncated versions of the protein showed that telomeric repeat binding factor 2 is involved in the processes of apoptosis and senescence (Karlseder et al., Science, 1999, 283, 1321-1325). While overexpression of the full-length telomeric repeat binding factor 2 failed to cause apoptosis, overexpression of a dominant negative form of the protein lacking both the N-terminal basic and the Myb DNA-binding domain resulted in rapid induction of apoptosis (Karlseder et al., Science, 1999, 283, 1321-1325). The effects seen were cell line dependent and did not occur in cell lines that lacked an apoptotic response to DNA damage or were deficient in p53, a tumor suppressor gene. This suggests that telomeric repeat binding factor 2 blocks an apoptotic pathway normally triggered upon DNA damage and that inhibiting telomeric repeat binding factor 2 function in tumorigenic cells may be of therapeutic value in these cell lines by antagonizing tumor progression.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of telomeric repeat binding factor 2. To date, strategies aimed at modulating telomeric repeat binding factor 2 function have involved the use of truncated forms of the protein that mimic the binding domains and block the DNA binding properties of the protein.
Disclosed in PCT application WO 97/08314 is the nucleotide sequence of the first telomeric repeat binding factor to be isolated, TRF1, and the protein encoded thereby. Also disclosed are claims to DNA or protein sequences which are degenerate variants encoding a TRF. In addition, expression vectors for the production of the protein and host cells that are transfected with these vectors are claimed as are antibodies to a TRF and ribozymes that cleave a TRF. Antisense nucleic acid molecules, both DNA and RNA in nature, that hybridize to a TRF mRNA initiation codon or any portion of a TRF mRNA are also claimed (De Lange, 1997). More recently disclosed, in PCT application WO 99/15662, are the nucleic acid sequence of telomeric repeat binding factors 1 and 2 and the isolated nucleic acid sequences that comprise and code for the various domains within these telomeric repeat binding factors. Also disclosed are the proteins and proteolytic fragments thereof (De Lange et al., 1999). Antisense oligonucleotides and ribozymes are generally disclosed.
The modular nature of the telomeric repeat binding factor protein, containing several distinct functional domains, has led to the investigation of altered or truncated forms of the protein as possible inhibitors. Truncated forms of the nucleic acid encoding telomeric repeat binding factors 1 and 2, are disclosed in PCT application WO 98/36066 as are vectors and host cells that express the truncated variants and methods to identify the heterodimers formed between the truncated forms and the native telomeric repeat binding factor proteins (De Lange et al., 1998).
There remains a long felt need for agents capable of effectively inhibiting telomeric repeat binding factor 2 function and antisense technology is emerging as an effective means for reducing the expression of specific gene products. Therefore, this technology may prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of telomeric repeat binding factor 2 expression. Consequently, the present invention provides compositions and methods for modulating telomeric repeat binding factor 2 (TRF2) expression.